It has long been known that urinary trypsin inhibitor (UTI) is present in human urine, and that its concentration in urine increases due to kidney diseases.
Piette et al report in The European Journal of Medicine Vol. 1, 5 Sep. 1992, that urinary trypsin inhibitory activity can be a useful marker, particularly in patients with fever of unknown origin and/or elevated erythrocyte sedimentation rate. Kuwajima et al report in Clinical Biochemistry, Vol. 23, April 1990, Pp. 167-171 that the automated assay of urinary trypsin inhibitor may be useful for the clinical diagnosis of acute phase response.
Accordingly, urinalysis for UTI is an important diagnostic tool. Such analytical techniques typically involve contacting the urine sample with a trypsin substrate attached to a chromophore at either arginine or lysine since these are the amino acids which are cleaved by trypsin. The concentration of urinary trypsin inhibitors in the urine sample is inversely proportional to the intensity of the colored response of the chromophore since urinary trypsin inhibitors inhibit trypsin activity according to their concentration in the fluid test sample. In Japanese Public Patent Disclosure Bulletin No. 10-70997, published on Mar. 17, 1998, there is described a method for measuring the degree of inhibition of trypsin activity in urine by mixing a urine sample, an enzyme sample containing trypsin and a buffer together with calcium in the amount of 0.15 μmol or more per μg of trypsin in the reaction fluid to a maximum of 100 μmol calcium per mL of urine sample. In addition, surfactants are used to assist in dissolving the trypsin substrate in its organic solvent. This technique is apparently designed to mask the interference caused by calcium present in urine samples by adding a large excess of calcium to the assay reagents.
This assay technique involves a liquid phase test for trypsin inhibitors in urine using excess calcium to cover up calcium interference and surfactants to dissolve the trypsin substrate. This technique is not suitable for a dry phase assay because the amount of buffer needed in such an assay to overcome the buffer in urine would precipitate in urine.